Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online May 10, 2007
Stem Cells Vol. 25 No. 8 August 2007, pp. 1985 -1994
doi:10.1634/stemcells.2006-0676; www.StemCells.com
© 2007 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2006-0676v1
2006-0676v2
25/8/1985    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasper, G.
Right arrow Articles by Duda, G. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasper, G.
Right arrow Articles by Duda, G. N.

TISSUE-SPECIFIC STEM CELLS

Matrix Metalloprotease Activity Is an Essential Link Between Mechanical Stimulus and Mesenchymal Stem Cell Behavior

Grit Kaspera,b, Juliane D. Glaesera,c, Sven Geisslera,b, Andrea Odea,b, Jens Tuischera, Georg Matziolisa, Carsten Perkaa, Georg N. Dudaa,b

aMusculoskeletal Research Center Berlin, Charité - Universitätsmedizin, Berlin, Germany;
bBerlin-Brandenburg Center for Regenerative Therapies, Charité - Universitätsmedizin, Berlin, Germany;
cFree University Berlin, Department of Biology, Chemistry, Pharmacy, Berlin, Germany

Key Words. Matrix metalloproteinases • Adult stem cells • Regeneration • Mechanical stimulation

Correspondence: Grit Kasper, Ph.D., Musculoskeletal Research Center Berlin, Charité - Universitätsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Telephone: ++49-30-450 615116; Fax: ++49-30-450 559969; e-mail: Grit.Kasper{at}charite.de

Received on October 23, 2006; accepted for publication on April 27, 2007.

First published online in STEM CELLS EXPRESS  May 10, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Progenitor cells are involved in the regeneration of the musculoskeletal system, which is known to be influenced by mechanical boundary conditions. Furthermore, matrix metalloproteases (MMPs) and tissue-specific inhibitors of metalloproteases (TIMPs) are crucial for matrix remodelling processes that occur during regeneration of bone and other tissues. This study has therefore investigated whether MMP activity affects mesenchymal stem cell (MSC) behavior and how MMP activity is influenced by the mechanical stimulation of these cells. Broad spectrum inhibition of MMPs altered the migration, proliferation, and osteogenic differentiation of MSCs. Expression analysis detected MMP-2, -3, -10, -11, -13, and -14, as well as TIMP-2, in MSCs at the mRNA and protein levels. Mechanical stimulation of MSCs led to an upregulation of their extracellular gelatinolytic activity, which was consistent with the increased protein levels seen for MMP-2, -3, -13, and TIMP-2. However, mRNA expression levels of MMPs/TIMPs showed no changes in response to mechanical stimulation, indicating an involvement of post-transcriptional regulatory processes such as alterations in MMP secretion or activation. One potential regulatory molecule might be the furin protease. Specific inhibition of MMP-2, -3, and -13 showed MMP-13 to be involved in osteogenic differentiation. The results of this study suggest that MSC function is controlled by MMP activity, which in turn is regulated by mechanical stimulation of cells. Thus, MMP/TIMP balance seems to play an essential role in transferring mechanical signals into MSC function.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Bone regeneration is characterized by extensive matrix remodelling involving cartilage formation, mineralization, and its replacement by immature woven bone, which is further remodelled to lamellar bone [1, 2]. These processes need to be regulated both spatially and temporally. Mesenchymal stem cells (MSCs) are thought to be crucial for bone regeneration, possibly acting via migration into the site of injury, proliferating and differentiating into functional cells of the mesenchymal lineage (e.g., chondrocytes and osteocytes) [2, 3]. MSCs have gained increasing attention in recent years due to their capability for self-renewal and their potential for multilineage differentiation [4]. In addition to the contribution of biological factors, bone regeneration is also influenced by mechanical boundary conditions [5–8]. However, just how mechanical stability and healing outcome are linked on the molecular level remains unclear. In this respect, it is interesting that the expression of members of the gene family of matrix metalloproteases (MMPs) by differentiated mesenchymal cells, such as osteoblasts and chondrocytes, seems to be regulated by mechanical loading conditions [9, 10].

MMPs represent a family of endoproteinases including at least 25 members that are divided according to their substrate specificity, sequence similarity, and domain organization into collagenases, gelatinases, stromelysins, membrane type, and other MMPs [11]. MMPs are able to cleave virtually all components of the extracellular matrix but also other substrates such as growth factor binding proteins or latent growth factors, thus regulating their bioavailability [9, 10]. Each MMP is characterized by a specific substrate spectrum and substrate affinity [11, 12]. This complex system is further regulated by the interaction of MMPs with four tissue-specific inhibitors of metalloproteases (TIMPs; TIMP-1 to TIMP-4). Thus, MMPs can have promoting as well as inhibitory effects on angiogenesis, proliferation, apoptosis, and invasion, depending upon the cell type, the MMP/TIMP balance, and the availability of substrates [13]. The profound importance of MMPs in matrix remodelling processes suggests a functional involvement of these enzymes in bone development and regeneration. Indeed, a stage-specific expression of several MMPs, such as MMP-2, -9, and -13, has been demonstrated during these processes [14, 15]. Furthermore, effects on bone morphology have been described for MMP-9, -13, and -14 knockout mice and for MMP-2 and -13 mutations in human individuals [15, 16]. Deletion of the MMP-9 gene in mice led to fractures with delayed healing or nonunion, caused by the persistent production of cartilage at the site of injury [17]. These complications seem to be due to a limited bioavailability of vascular endothelial growth factor (VEGF) caused by the lack of MMP-9 activity. In addition, it is speculated that this protease is involved in the regulation of cell differentiation into the chondrogenic or osteogenic lineage [17]. It has been further hypothesized that additional MMP family members might play a role in MSC differentiation [18]. However, direct evidence remains scarce, and other functional characteristics of MSC biology have not yet been examined with regard to MMP activity.

In this study, we aim to determine the consequences of MMP inhibition on the functional behavior of MSCs. To elucidate which MMP family members might play a role for MSC function during musculoskeletal regeneration processes, the expression profile of MMPs/TIMPs in MSCs with and without mechanical loading was investigated in a bioreactor system for modeling conditions in a fracture gap during the early phase of bone healing [19, 20].


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Cell Culture
Bone marrow was obtained from human donors undergoing hip surgery. All donors gave informed consent. MSCs were isolated by density separation using Histopaque-1077 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and subsequent plastic adherence. The mean donor age was 67 years (ranging 45–84 years). Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, http://www.invitrogen.com) supplemented with 10% fetal calf serum (FCS) (Biochrom AG, Berlin, http://www.biochrom.de) and 10 U/ml penicillin plus 100 µg/ml streptomycin was used as expansion medium for MSCs. Cells were passaged at 70%–80% confluence and passages 3–7 were used for experiments. A homogeneous population of MSCs was determined by fluorescence-activated cell sorting analysis of the surface markers CD73+, CD44+, CD105+, CD106+, D7fib+, CD34–, CD4–, and CD45–. Their capacity to differentiate into the osteogenic, adipogenic, and chondrogenic lineage by addition of appropriate media [3] was also confirmed. For chondrogenic differentiation, a pellet culture with chondrogenic medium was employed [21].

Functional Assays
For functional assays with broad spectrum MMP inhibitors, GM6001 (4 µg/ml), TIMP-2 (200 ng/ml; both Chemicon, Temecula, CA, http://www.chemicon.com), and minocycline (250 µg/ml; Calbiochem, San Diego, http://www.emdbiosciences.com) were used. To analyze the influence of MMPs for MSC function specifically, a MMP-2 inhibitor (2 µg/ml) (Calbiochem), a MMP-3 inhibitor (3.16 µg/ml) (Calbiochem), and a MMP-13 inhibitor CL-82198 (33.9 µg/ml) (Calbiochem, Germany) were employed in all assays. CL-82198 is thought not to be active against other MMPs with similar catalytic sites, such as MMP-1 and MMP-9 [22]. The MMP-2 inhibitor seems to additionally inhibit MMP-9 (MMP-9 expression appears to be absent in the employed cells; see Results). Furthermore, Ki values for gelatinase inhibition are 35-fold lower than for the collagenase MMP-1 [23]. An interaction of the potent MMP-3 inhibitor with other MMPs has, however, so far not been excluded [24]. Tissue culture plastic and filters were coated with matrigel (1 mg/ml; BD Biosciences, San Diego, http://www.bdbiosciences.com) or collagen (100 µl/ml; Sigma) overnight at 4°C. Migration of MSCs was measured using a modified Boyden chamber assay [25], for which coated polycarbonate filters (8 µm pore size; Nunc, Rochester, NY, http://www.nuncbrand.com) were used in 24-well plates. Five hundred microliters of expansion medium containing the aforementioned inhibitors was placed in each of the lower chambers. We seeded 3 x 104 MSCs onto the filters in 500 µl of expansion medium supplemented with inhibitor. After a 5-hour incubation period at 37°C, cells were fixed by paraformaldehyde. After removing cells from the upper side of the filter by scraping, migrated cells were stained with 10 µg/ml Hoechst (Molecular Probes, Eugene, OR, http://probes.invitrogen.com). The number of migrated cells was counted in five microscope regions per filter for two filters per sample. To analyze proliferation, 1.3 x 103 MSCs were seeded onto coated 96-well plates in DMEM supplemented with 5% FCS and appropriate substances. The CellTiter 96 AQueous test (Promega, Madison, WI, http://www.promega.com) was conducted after 4 days of incubation, with assays carried out in triplicates. To measure differentiation, 4 x 103 MSCs were cultured on coated 96 well plates. Cells were exposed to osteogenic medium for 2 weeks after reaching 80% confluence. Osteogenic differentiation was detected by alkaline phosphatase (AP) staining using para-nitrophenylphosphate and Alizarin red (AR) staining (both Sigma). All values were then normalized to cell number, as determined using the 96 AQueous test. Five replications were performed per sample, with all functional assays carried out as three independent experiments.

Bioreactor Experiments
A previously described bioreactor system was employed [19]. Briefly, MSCs were trypsinized, and 2 x 106 cells were resuspended in 350 µl of expansion medium. This suspension was then mixed with 300 µl of fibrinogen/medium (1:2) (Tissucol; Baxter, Deerfield, IL http://www.baxter.com) and 50 µl of thrombin S/medium (1:2). The mixture was allowed to solidify for 30 minutes at 37°C. The construct was sandwiched between two cancellous bone chips and placed into the bioreactor. Twenty-five ml of MSC expansion medium was added containing 0.6 ml Trasylol (Bayer, Leverkusen, Germany, http://www.bayer.com). A pressure of 10 kPa at a frequency of 1 Hz, which produced approximately 30% strain, was applied in accordance with in vivo measurements of intrafragmentary movement [20]. Mechanical loading was carried out for 72 hours. Afterward, the conditioned medium (CM) was harvested. To investigate the involvement of furin proteases, medium was supplemented with a furin inhibitor I (4 µg/ml) (Calbiochem) during mechanical stimulation. For Western blot analysis, the medium was replaced by expansion medium without FCS and conditioned for 8 hours. Equal cell numbers in constructs exposed to mechanical loading and unloaded controls were validated by means of the 96 AQueous test.

RNA Expression Analysis
RNA was isolated by the RNeasy Mini kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) as instructed by the manufacturer. RNA integrity was assessed by means of the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, http://www.agilent.com). Expression analysis was carried out by the GEArray Q Series Kit for Human Extracellular Matrix and Adhesion Molecules (SuperArray Bioscience Corporation, Frederick, MD, http://www.superarray.com) according to the manufacturer's protocol. Then, 1–3 µg RNA were isolated from loaded and unloaded bioreactor constructs, reverse transcribed to Biotin-16-UTP labeled cDNA, and separately hybridized onto gene chips. Signals were detected by chemiluminescence. For analyzing gene spot intensities of scanned x-ray films, the program ScanAlyze (version 2.5; Eisen Lab, Berkeley, CA, http://rana.lbl.gov/EisenSoftware.htm) was employed. Here, the GEArray Analyzer software (v 1.3; SuperArray) was used for background correction. Each array experiment was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (as internal standard). A detection limit was set at 0.05 times the GAPDH signal. Data from four donors were determined in independent experiments.

Zymography, Enzyme-Linked Immunosorbent Assay, and Western Blotting
Gelatin zymography was performed on 10% Novex zymogram gels (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to manufacturer's protocol, employing 20 µl of CM (1:30 diluted for MMP-2 detection). Gelatinolytic activity of MMP-2 in CM was quantified by a serial dilution of active human recombinant MMP-2 (Calbiochem). For standardization, CM corresponding to equal cell numbers was applied to the gels. Analysis of the digital images was carried out using the NIH Image J software package (http://rsb.info.nih.gov/nih-image). Enzyme-linked immunosorbent assays (ELISAs) (polyclonal antibodies for MMP-2, -3, TIMP-2 and monoclonal antibody for MMP-10 from R&D Systems [Minneapolis, http://www.rndsystems.com] and monoclonal MMP-13 from Amersham Biosciences [Piscataway, NJ, http://www.amersham.com]) were performed according to manufacturer's instructions using triplicates. CM was applied as follows: MMP-2 and TIMP-2, undiluted; MMP-10, 1:5 concentrated; MMP-3 and -13, 1:20 concentrated. CM was concentrated by Amicon Ultra 5K Centrifugal Filter Devices (Millipore, Billerica, MA, http://www.millipore.com).

Whole-cell lysates were generated by resuspending cells in SDS lysis buffer (30 mM Tris-HCL pH 6.8, 10% Glycerol, 1% SDS, 1 mM EDTA pH 8) supplemented with Proteinase Inhibitor Cocktail Complete Mini (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). For Western blot analysis, the Novex system (Invitrogen) was employed according to manufacturer's instructions. We separated 1:20 concentrated CM and 10 µg of cell lysate on 4%–12% NuPAGE gradient gels. The primary antibodies (Ab) used were rabbit({alpha}-human MMP-3) (Ab-5, 1:50), rabbit({alpha}-human MMP-10) (1:100), rabbit({alpha}-human MMP-13) (Ab-5, 1:100), rabbit({alpha}-human MMP14) (Ab-1, 1:100), rabbit({alpha}-human MMP-2) (1:50), rabbit({alpha}-human TIMP-2) (Ab-7, 1:100), and mouse({alpha}-human MMP-11) (Ab-5 clone SL3.05, 1:250) (all from Neomarkers, Fremont, CA, http://www.labvision.com). The secondary antibodies used were donkey({alpha}-rabbit IgG)peroxidase (Amersham; 1:1,000 for MMP-10, 1:5,000 for MMP-13, 1:6,000 for MMP-2 and -14, and 1:8,000 for MMP-3) and goat({alpha}-mouse IgG)peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com; 1:10,000). Signals were detected by means of ECL Plus (Amersham). As positive controls, CM and lysates from the cell lines MDA-MB-231, MG-63, or commercially available positive controls (Neomarkers) were used. Data were determined from at least four donors for zymography experiments, at least six donors for ELISAs, and at least three donors for Western blot analysis.

Statistics
For statistical evaluation, the software package SPSS 12.0 was used. Results from functional assays were analyzed for statistical significance using Student's t test. Data from expression analyses were tested by the Wilcoxon's test. Two-sided tests, with a significance level of p < .05, were employed for all experiments.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
Functional Analysis with Broad Spectrum MMP Inhibitors
In order to examine the relevance of MMPs for MSC behavior, the three broad spectrum MMP inhibitors GM6001, minocycline, and TIMP-2 were employed in functional assays for migration, proliferation, and osteogenic differentiation of human MSCs (Fig. 1). Different coatings of tissue culture plastic or migration filters were used to simulate a complex extracellular matrix environment (matrigel) and a defined environment resembling bone (collagen). Cell migration was diminished in the presence of MMP inhibitors (Fig. 1A). Minocycline and TIMP-2 significantly reduced migration on collagen-coated filters (minocycline: mean = 58%, .001 < p < .014; TIMP-2: mean = 74%, .001 < p < .003). On matrigel-coated filters, migration tended to be decreased by GM6001, minocycline, and TIMP-2, but no statistical significance was reached. Also, the proliferative capacity of MSCs was influenced by MMP inhibition (Fig. 1B). On collagen-coated plates, all three inhibitors resulted in a significant decrease of MSC proliferation (GM6001: mean = 71%, .011 < p < .046; minocycline: mean = 60%, .001 < p < .014; TIMP-2: mean = 64%, .009 < p < .023). On matrigel-coated plates, minocycline significantly reduced proliferation (mean = 55%, p < .001). Osteogenic differentiation of MSCs was determined by their ability to deposit mineralized matrix and by their AP activity (Fig. 1C). In medium providing an osteogenic stimulus, both parameters were diminished in the presence of minocycline on collagen as well as on matrigel-coated plates (AR: meanmatrigel = 11%, .001 < pmatrigel < .033; meancollagen = 8%, pcollagen < .001; AP: meanmatrigel = 55%, .001 < pmatrigel < .015; meancollagen = 36%, .001 < pcollagen < .019). However, in standard expansion medium for MSCs, no significant effect of the supplemented MMP inhibitors was observed.


Figure 1
View larger version (34K):
[in this window]
[in a new window]

  		Figure 1. Matrix metalloprotease (MMP) activity is involved in migration, proliferation, and differentiation of MSCs. (A): Photographs show two representative filters with migrated cells investigated in a modified Boyden chamber assay. Cell nuclei were stained by Hoechst. The diagram displays numbers of migrated cells relative to controls (pcollagen, minocycline < .014; pcollagen, TIMP-2 < .003). (B): Typical wells in proliferation assays. Cell numbers are shown in relation to controls in the diagram (pcollagen, GM6001 < .046; pcollagen, minocycline < .014; pcollagen, TIMP-2 < .023; pmatrigel, minocycline < .001). (C): Photographs present AR staining of two typical wells in differentiation assays. Osteogenic differentiation determined by AR and AP staining was evaluated in relation to control samples and is displayed in the diagram (AR: pmatrigel, minocycline < .033, pcollagen, minocycline < .001; AP: pmatrigel, minocycline < .015, pcollagen, minocycline < .019). Assays were carried out in the presence of the MMP inhibitors GM6001, minocycline, and TIMP-2 on collagen- and matrigel-coated plates. Results were normalized to vehicle controls; * indicates statistical significance. Number of experiments: n = 3. Abbreviations: AP, alkaline phosphatase; AR, Alizarin red; neg., negative; TIMP, tissue-specific inhibitor of metalloproteases.

 
Expression Analysis of MMPs/TIMPs in MSCs
Subsequently, the question as to which MMPs and TIMPs are expressed by MSCs and could therefore be responsible for the observed effects of MMP inhibition was addressed. Hybridization of SuperArray gene chips revealed that the collagenase MMP-13, the gelatinase MMP-2, and the inhibitor TIMP-2 are expressed at relatively high levels in MSCs (relative to GAPDH expression levels: medianMMP-13 = 0.54, medianMMP-2 = 0.80, medianTIMP-2 = 0.34; Fig. 2A). For MMP-11, -14, -9, and -20 and TIMP-1 and -3, expression levels were much lower and reached the applied detection limit in cells from some but not all donors. Not detectable were the stromelysins MMP-3 and -10, the collagenases MMP-1 and -8, membrane anchored MMP-15, -16, -17, and -24, the matrilysins MMP-7 and -26, and the unclassified MMP-12. To verify the mRNA expression pattern of secreted MMPs at the protein level, CM from MSCs was investigated by Western blot analysis (Fig. 2B). Obtained results revealed differences between mRNA and protein expression pattern. In detail, all stromelysins (MMP -3, -10, -11), collagenase MMP-13, and gelatinase MMP-2, as well as the inhibitor TIMP-2, were secreted by MSCs, although no mRNA was detected for MMP-3 and -10. This is likely due to the specification of the detection limit in mRNA measurement. The specificity of antibodies was demonstrated by accurate prediction of molecular masses in Western blots and the usage of monoclonal antibodies in ELISAs for MMP-10 detection. Furthermore, membrane-associated MMP-14 was clearly present in MSC cell lysates, although mRNA expression levels were very low or did not reach the detection limit. In contrast, at the protein level, MMP-9 and -20 and TIMP-1 and -3 were not detectable (data not shown). Again, the applicability of antibodies was proved in positive controls. Furthermore, the employment of monoclonal antibodies in ELISAs confirmed the results for TIMP-1 and MMP-9.


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		Figure 2. MMP/TIMP mRNA and protein expression pattern of MSCs. (A): Results from gene array hybridization conducted with RNA lysates from MSCs. Signal intensities of MMP/TIMP transcripts are displayed after normalization to GAPDH. Bars represent median values. Digits indicate the number of donors with the corresponding value. (B): Western blot analysis of CM and whole cell lysates of MSCs are displayed. Arrows indicate MMP/TIMP expression by MSCs. As positive controls, the cell lines MDA-MB-231 and MG-63 and purchasable positive controls (control) were used. Negative controls were not treated with primary antibodies. Abbreviations: CM, conditioned medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, goat({alpha}-mouse IgG)peroxidase; MMP, matrix metalloprotease; neg., negative; R, donkey({alpha}-rabbit IgG)peroxidase; TIMP, tissue-specific inhibitor of metalloproteases.

 
Mechanically Regulated MMPs and TIMPs
In order to investigate which MMPs are likely to play a role in regeneration processes of the musculoskeletal system, a bioreactor system that aims to resemble the initial conditions during the early phase of bone healing was employed [19]. Analysis of the mRNA levels of the abundantly expressed transcripts MMP-13, -2, and TIMP-2 showed no differential expression between mechanically loaded MSCs and unloaded controls (supplemental online Table 1). To examine the extracellular gelatinolytic activity resulting from secreted MMPs by MSCs in response to mechanical loading, zymography of the CM was conducted (Fig. 3A). Comparison to known amounts of recombinant MMP-2 in zymography revealed that concentrations of active MMP-2 in CM from loaded MSCs are in the range of 74 ng/ml and 148 ng/ml detected in MSCs from three donors (due to the detection limit of the employed software, data were determined previous to FCS background subtraction). Quantification of MMP-2 concentrations by ELISA resulted in more precise protein amounts (medianloaded MMP-2: 54.77 ng/ml, range: 33.72 ng/ml to 102.17 ng/ml). MMP-3 and MMP-13 were present at much lower concentrations (medianloaded MMP-3: 12 pg/ml, range: 8 pg/ml to 3.33 ng/ml; meanloaded MMP-13: 20 pg/ml, range: 6 pg/ml to 50 pg/ml, both determined by ELISA). Results showed a clear enhancement of total gelatinolysis in mechanically stimulated MSCs (median = 160%, p = .036; Fig. 3B). CM from negative controls (constructs without cells) showed a background gelatinolytic activity, most likely resulting from the applied FCS, but no increase in response to mechanical loading (meanneg control = 100%; pneg control = .686). In particular, gelatinase activities at molecular weights corresponding to MMP-13 and -2 and an unknown gelatinase were significantly increased in response to mechanical loading, which was not observed in control constructs without cells (MMP-13: median = 130%, p = .043; MMP-2: median = 150%, p = .008; unknown gelatinase: median = 220%, p = .012). By employing commercially available ELISAs, the observed upregulation of MMP-13 and -2 was confirmed (MMP-13: median = 280%, p = .028; MMP-2: median = 150%, p = .004; Fig. 3C). Furthermore, it was shown that MMP-3 and TIMP-2 are upregulated in CM of mechanically loaded MSCs (MMP-3: median = 170%, p = .028; TIMP-2: median = 140%, p = .011; Fig. 3C). MMP-10 showed a tendency for differential expression, which did not reach statistical significance (MMP10: median = 150%, p = .060). These effects were not detected in negative control constructs.


Figure 3
View larger version (38K):
[in this window]
[in a new window]

  		Figure 3. Gelatinolytic activity and MMP amounts are increased by mechanical stimulation. (A): Representative zymogram showing gelatinolytic activity of MSCs with and without mechanical stimulation. (B): Results of the quantification of signal intensities of total gelatinolytic activity as well as molecular weight of MMP-13, -2, and an unknown gelatinase are displayed. For quantification of MMP-2 signal, conditioned media (CM) were diluted 1:30 before zymography (ptotal gelatinolysis = .036; pMMP-13 = .043; pMMP-2 = .008; punknown gelatinase = .012). (C): Results from enzyme-linked immunosorbent assay (ELISA) experiments specific for MMP-3, -10, -13, -2, and TIMP-2 are shown. Protein levels in CM of mechanically stimulated MSCs were normalized to values from CM of control constructs that were not exposed to mechanical stimulation. As a negative control, intensities of CM from loaded and unloaded constructs without cells were determined (pMMP-13 = .028; pMMP-2 = .004; pMMP-3 = .028; pTIMP-2 = .011). * indicates statistical significance. Extreme values are presented as {blacktriangleup} and outliers as bullet. An extreme value for MMP-13 (normalized activity 1,130%) is not displayed in the zymogram. Number of experiments: nzymography = 5; nELISA = 6. Abbreviations: MMP, matrix metalloprotease; neg., negative; TIMP, tissue-specific inhibitor of metalloproteases; w/o, without.

 
Role of Furin in MMP-2 Upregulation
To investigate the contribution of furin to the mechanical regulation of MMP-2, the medium was supplemented with a furin inhibitor during mechanical stimulation. Zymogram analysis showed a trend of suppression of the MMP-2 induction caused by mechanical loading, which did not reach the statistical significance level (factor loaded normalized to unloaded: meanfurin_inhibitor(dimethyl-sulfoxide [DMSO]), 1; meancontrol(DMSO), 1.6; p = .068; Figure 4). Negative controls without cells in the construct and supplementation of furin inhibitor showed no differences in MMP-2 activity.


Figure 4
View larger version (28K):
[in this window]
[in a new window]

  		Figure 4. Possible involvement of furin in MMP-2 upregulation. Quantification of signal intensities of MMP-2 detected in conditioned medium (CM) supplemented with furin inhibitor is shown. Protein levels in CM of mechanically stimulated MSCs were normalized to values from CM of control constructs that were not exposed to mechanical stimulation. CM from loaded and unloaded constructs without inhibitor was used as positive control. As a negative control, intensities of CM from loaded and unloaded constructs without cells were determined (pfurin inhibitor = .068). Number of experiments: n = 4. Abbreviations: MMP, matrix metalloprotease; w/o, without.

 
Functional Analysis with MMP Specific Inhibitors
To determine whether the identified mechanically regulated MMPs are involved in migration, proliferation, and osteogenic differentiation of MSCs, functional assays were conducted in the presence of MMP-2, MMP-3, and MMP-13 specific inhibitors. Cell migration tended to be decreased in the presence of the MMP-2 inhibitor, but no statistical significance was reached (Fig. 5A). No altered proliferation was detected in the presence of these inhibitors (Fig. 5B). MMP-13 was shown to be involved in osteogenic differentiation of MSCs (Fig. 5C). On matrigel-coated plates, AP activity was significantly diminished in the presence of the MMP-13 inhibitor in osteogenic as well as in expansion media (APMMP-13: meanosteogenic medium = 60%, .001 < p < .013; meanexpansion medium = 72%, .001 < p < .014). Also, the ability to deposit mineralized matrix tended to be reduced by MMP-13 inhibition, but no statistical significance was reached. On collagen-coated plates, matrix mineralization tended to decrease in response to MMP-13 inhibition in osteogenic medium and to increase by MMP-2 inhibition in expansion medium. However, no statistical significance was reached.


Figure 5
View larger version (21K):
[in this window]
[in a new window]

  		Figure 5. MMP-13 is involved in the osteogenic differentiation of MSCs. (A): The numbers of (A) migrated cells and (B) proliferated cells are not affected by MMP inhibition. (C): Osteogenic differentiation is significantly reduced in the presence of an MMP-13 inhibitor. Assays were carried out in the presence of the MMP-2, -3, and -13 inhibitors. Results were normalized to vehicle controls (APmatrigel: pMMP-13, osteogenic medium < .013, pMMP-13, expansion medium < .014); * indicates statistical significance. Number of experiments: n = 3. Abbreviations: AP, alkaline phosphatase; AR, Alizarin red; MMP, matrix metalloprotease.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 
This study has demonstrated the regulation of MSC behavior by MMPs, a process that is in accordance with postulations that migration and differentiation of MSCs involve MMP activity [18, 26]. Another relevant finding has been the upregulation of extracellular MMP-2, -3, -13 and TIMP-2 in response to mechanical loading. Specific inhibition of the mechanically regulated MMPs demonstrated the involvement of MMP-13 in the osteogenic differentiation of MSCs. This could be associated with the growth plate defects seen in MMP-13 knockout mice and in human individuals possessing mutations in the MMP-13 gene [16, 27]. Not all of the effects mediated by broad spectrum MMP inhibition, however, were observed when single MMPs were inhibited. This might indicate the involvement of additional, possibly mechanically insensitive MMPs. Furthermore, the effects of single MMP inhibition might be dependent upon the activity of other family members, for example, MMPs that could act synergistically as observed in MMP-9/MMP-13 knockout animals [16] or other MMP family members that could compensate for lost activity [28].

Comparison of MMP mRNA and protein expression levels has revealed that they are not strictly correlated. According to a broader investigation, however, the discrepancy between mRNA and protein levels seems to be more a rule than an exception [29]. This is especially important, since most former expression analyses of MMPs in MSCs were conducted at the transcriptional level [26, 30, 31]. Furthermore, MMP-2, -3, -13 and TIMP-2 protein but not mRNA levels were shown to be specifically upregulated by mechanical loading of MSCs. These findings indicate the involvement of post-transcriptional regulatory processes in response to mechanical stimulation, instead of a direct activation of transcription, for example, by mechanosensitive promoter elements [32, 33]. Indeed, furin activity might be involved in the elevation of extracellular levels of active MMP-2. Since the intracellular protease furin is able to activate MMP-14, which in turn generates active MMP-2 [34], one could speculate that the effect of mechanical loading on extracellular MMP-2 levels is based on the regulation of the furin/MMP-14 axis. In general, MMPs could be directly or indirectly regulated by growth factors that respond to changes in the mechanical environment. Candidates for such factors are transforming growth factor (TGF)-ß1, interleukin-1ß, and tumor necrosis factor-{alpha}, since they have been shown to control MMP expression in MSCs or MSC-derived cells [35]. In addition, post-translational mechanisms such as cleavage of MMP proenzymes and modulation of the secretory pathway or of protein stability seem to regulate MMP protein levels [36, 37, 38]. Furthermore, post-transcriptional modifications could be responsible for altered MMP protein levels while mRNA levels remain constant [39, 40]. In contrast to the results presented in this study, other investigations using different experimental settings demonstrated changes in the amounts of MMP mRNA by mechanical loading [41, 42], indicating the existence of additional regulatory mechanisms on mRNA level in other mesenchymal cells. Furthermore, the nature of the applied stress seems to be important for the regulation of matrix remodelling by MSCs [43, 44].

The enhanced gelatinolytic activity of mechanically stimulated MSCs, as demonstrated in this study, could have manifold effects, possibly contributing to tissue remodelling processes such as cartilage removal and its replacement by woven bone [15]. Alterations to MMP activity, for example, could lead to the degradation of extracellular matrix components like collagen, thereby removing mechanical barriers or exposing cryptic sites that could act to regulate MSCs [45, 46]. Furthermore, the modulation of growth factor activity or bioavailability is another likely mechanism of MMP impact on MSC behavior [9]. Indeed, MMPs have been shown to modulate molecules important for MSC migration, differentiation, and proliferation such as TGF-ß, VEGF, or fibroblast growth factor [47–49]. The pro-osteogenic bone morphogenic proteins might be a further candidate target of MMP activity, since MMPs are known to efficiently make members of the TGF-ß family available by displacing them from the extracellular matrix [45]. In addition to a modified proteolytic activity, MSCs seem to respond to cyclic mechanical stress by an altered synthesis of matrix components, thereby further influencing their microenvironment [44].

Based on the data obtained in this study, a mechanism for translation of mechanical signals into cellular responses is postulated (Fig. 6). Mechanical boundary conditions seem to directly determine the MMP/TIMP expression pattern of MSCs. Alteration of the MMP/TIMP balance could lead to an enhancement of proteolytic and especially gelatinolytic activity of MSCs. This might be especially important for bone, where collagen is the major extracellular matrix component. This activity could create regulatory signals such as changes in mechanical conditions due to the plain breakdown of extracellular matrix components or could result in generation of regulatory extracellular matrix cleavage products and (in)activation of signaling molecules such as growth factors or receptors. Thereby, a microenvironment for MSCs might be created in an autocrine manner, which may determine the functional behavior, such as migration, proliferation, and differentiation of these cells. In addition, paracrine consequences could further regulate angiogenesis [50] or lead to the recruitment of other progenitor cells, such as hematopoietic stem cells or osteoclast precursors [51, 52].


Figure 6
View larger version (32K):
[in this window]
[in a new window]

  		Figure 6. MMP/TIMP balance as a potential transducer of mechanical stimuli into MSC cellular functions. From the obtained data, the following mechanism can be hypothesized: mechanical loading of MSCs leads to enhanced secretion and, potentially, activation of MMPs/TIMPs (a), which mediate different regulatory processes (b), such as degradation of the ECM (c) or release of latent growth factors (d). Interaction of ECM fragments or activated growth factors with cell surface receptors (e) results in altered MSC migration, proliferation, and differentiation in an autocrine/paracrine manner. Abbreviations: ECM, extracellular matrix; MMP, matrix metalloprotease; TIMP, tissue-specific inhibitor of metalloproteases.

 
However, the physiological relevance of such in vitro results needs to be considered cautiously, since it is known that mesenchymal cells, such as osteoblasts, chondrocytes, and adipocytes, are able to dedifferentiate during in vitro cultivation into less committed precursor cells [53, 54]. Therefore, in vitro expanded MSCs might represent dedifferentiated stroma cells.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosure of Potential...
 Acknowledgments
 References
 
Mechanical stimuli have been shown to influence mesenchymal cell characteristics such as proliferation and differentiation [55–57]. From the results of this study, it seems likely that the translation of these mechanical stimuli into the cellular response of MSCs might be mediated by the regulation of MMPs. These regulatory mechanisms could be important for keeping MSCs in a quiescent state in their physiological stem cell niche [4] and for activating these cells in the case of injury, for example, by MMP inducing stimuli in response to mechanical forces present in a fracture site. Since several different MMPs are likely to be affected, further investigations should evaluate the effect of complex alterations in the MMP/TIMP balance rather than the separate investigation of single MMP members. Understanding the mechanisms of MMP involvement in MSC biology and regeneration/developmental processes is not only important for potentially novel treatment strategies in bone healing but also for assessing unintentional side effects of pharmacological inhibition of MMP activity.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
References
 
This study was partially supported by the Bundesministerium für Bildung und Forschung excellence cluster Berlin-Brandenburg Center of Regenerative Medicine and the Arbeitsgemeinschaft Osteosynthese Foundation. The authors are grateful to M. Tschirschmann for help with RNA analysis and M. Princ for excellent technical assistance. G.K. and J.D.G. contributed equally to this work.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Probst A, Spiegel HU. Cellular mechanisms of bone repair. J Invest Surg 1997;10:77–86.[Medline]

  2. Einhorn TA. The cell and molecular biology of fracture healing. Clin Orthop Relat Res 1998; (suppl 355)S7–S21.

  3. Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.[Abstract/Free Full Text]

  4. Baksh D, Song L, Tuan RS. Adult mesenchymal stem cells: Characterization, differentiation, and application in cell and gene therapy. J Cell Mol Med 2004;8:301–316.[Medline]

  5. Duda GN, Sporrer S, Sollmann M et al. Interfragmentary movements in the early phase of healing in distraction and correction osteotomies stabilized with ring fixators. Langenbecks Arch Surg 2003;387:433–440.[Medline]

  6. Klein P, Opitz M, Schell H et al. Comparison of unreamed nailing and external fixation of tibial diastases—mechanical conditions during healing and biological outcome. J Orthop Res 2004;22:1072–1078.[CrossRef][Medline]

  7. Klein P, Schell H, Streitparth F et al. The initial phase of fracture healing is specifically sensitive to mechanical conditions. J Orthop Res 2003;21:662–669.[CrossRef][Medline]

  8. Lienau J, Schell H, Duda GN et al. Initial vascularization and tissue differentiation are influenced by fixation stability. J Orthop Res 2005;23:639–645.[CrossRef][Medline]

  9. Sternlicht MD, Werb Z. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol 2001;17:463–516.[CrossRef][Medline]

  10. Chang C, Werb Z. The many faces of metalloproteases: Cell growth, invasion, angiogenesis and metastasis. Trends Cell Biol 2001;11:S37–S43.[Medline]

  11. Stamenkovic I. Matrix metalloproteinases in tumor invasion and metastasis. Semin Cancer Biol 2000;10:415–433.[CrossRef][Medline]

  12. Overall CM. Molecular determinants of metalloproteinase substrate specificity: Matrix metalloproteinase substrate binding domains, modules, and exosites. Mol Biotechnol 2002;22:51–86.[CrossRef][Medline]

  13. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2002;2:161–174.[Medline]

  14. Gerstenfeld LC, Cullinane DM, Barnes GL et al. Fracture healing as a post-natal developmental process: Molecular, spatial, and temporal aspects of its regulation. J Cell Biochem 2003;88:873–884.[CrossRef][Medline]

  15. Ortega N, Behonick DJ, Werb Z. Matrix remodeling during endochondral ossification. Trends Cell Biol 2004;14:86–93.[CrossRef][Medline]

  16. Stickens D, Behonick DJ, Ortega N et al. Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development 2004;131:5883–5895.[Abstract/Free Full Text]

  17. Colnot C, Thompson Z, Miclau T et al. Altered fracture repair in the absence of MMP9. Development 2003;130:4123–4133.[Abstract/Free Full Text]

  18. Mannello F, Tonti GA, Bagnara GP et al. Role and function of matrix metalloproteinases in the differentiation and biological characterization of mesenchymal stem cells. STEM CELLS 2006;24:475–481.[Abstract/Free Full Text]

  19. Matziolis G, Tuischer J, Kasper G et al. Simulation of cell differentiation in fracture healing-mechanically loaded composite scaffolds in a novel bioreactor system. Tissue Eng 2006;12:201–208.[CrossRef][Medline]

  20. Claes LE, Heigele CA, Neidlinger-Wilke C et al. Effects of mechanical factors on the fracture healing process. Clin Orthop Relat Res 1998; (suppl 355)S132–S147.

  21. Sekiya I, Vuoristo JT, Larson BL et al. In vitro cartilage formation by human adult stem cells from bone marrow stroma defines the sequence of cellular and molecular events during chondrogenesis. Proc Natl Acad Sci U S A 2002;99:4397–4402.[Abstract/Free Full Text]

  22. Chen JM, Nelson FC, Levin JI et al. Structure-based design of a novel, potent, and selective inhibitor for MMP-13 utilizing NMR spectroscopy and computer-aided molecular design. J Am Chem Soc 2000;122:9648.[CrossRef]

  23. Berton A, Rigot V, Huet E et al. Involvement of fibronectin type II repeats in the efficient inhibition of gelatinases A and B by long-chain unsaturated fatty acids. J Biol Chem 2001;276:20458–20465.[Abstract/Free Full Text]

  24. MacPherson LJ, Bayburt EK, Capparelli MP et al. Discovery of CGS 27023A, a non-peptidic, potent, and orally active stromelysin inhibitor that blocks cartilage degradation in rabbits. J Med Chem 1997;40:2525–2532.[CrossRef][Medline]

  25. Falk W, Goodwin RH Jr., Leonard EJ. A 48-well micro chemotaxis assembly for rapid and accurate measurement of leukocyte migration. J Immunol Methods 1980;33:239–247.[CrossRef][Medline]

  26. Son BR, Marquez-Curtis LA, Kucia M et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by SDF-1-CXCR4 and HGF-c-met axes and involves matrix metalloproteinases. STEM CELLS 2006;24:1254–1264.[Abstract/Free Full Text]

  27. Kennedy AM, Inada M, Krane SM et al. MMP13 mutation causes spondyloepimetaphyseal dysplasia, Missouri type (SEMD(MO). J Clin Invest 2005;115:2832–2842.[CrossRef][Medline]

  28. Page-McCaw A, Ewald AJ, Werb Z. Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 2007;8:221–233.[CrossRef][Medline]

  29. Gygi SP, Rochon Y, Franza BR et al. Correlation between protein and mRNA abundance in yeast. Mol Cell Biol 1999;19:1720–1730.[Abstract/Free Full Text]

  30. Silva WA Jr., Covas DT, Panepucci RA et al. The profile of gene expression of human marrow mesenchymal stem cells. STEM CELLS 2003;21:661–669.[Abstract/Free Full Text]

  31. Panepucci RA, Siufi JL, Silva WA Jr. et al. Comparison of gene expression of umbilical cord vein and bone marrow-derived mesenchymal stem cells. STEM CELLS 2004;22:1263–1278.[Abstract/Free Full Text]

  32. Chakraborti S, Mandal M, Das S et al. Regulation of matrix metalloproteinases: An overview. Mol Cell Biochem 2003;253:269–285.[CrossRef][Medline]

  33. Chiquet M. Regulation of extracellular matrix gene expression by mechanical stress. Matrix Biol 1999;18:417–426.[CrossRef][Medline]

  34. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: Structure, function, and biochemistry. Circ Res 2003;92:827–839.[Abstract/Free Full Text]

  35. Ries C, Egea V, Karow M et al. MMP-2, MT1-MMP, and TIMP-2 are essential for the invasive capacity of human mesenchymal stem cells: Differential regulation by inflammatory cytokines. Blood 2007;109:4055–4063.[Abstract/Free Full Text]

  36. Sehgal I, Thompson TC. Novel regulation of type IV collagenase (matrix metalloproteinase-9 and -2) activities by transforming growth factor-beta1 in human prostate cancer cell lines. Mol Biol Cell 1999;10:407–416.[Abstract/Free Full Text]

  37. Kjeldsen L, Sengelov H, Lollike K et al. Isolation and characterization of gelatinase granules from human neutrophils. Blood 1994;83:1640–1649.[Abstract/Free Full Text]

  38. Taraboletti G, Sonzogni L, Vergani V et al. Posttranscriptional stimulation of endothelial cell matrix metalloproteinases 2 and 1 by endothelioma cells. Exp Cell Res 2000;258:384–394.[CrossRef][Medline]

  39. Bradley JM, Kelley MJ, Rose A et al. Signaling pathways used in trabecular matrix metalloproteinase response to mechanical stretch. Invest Ophthalmol Vis Sci 2003;44:5174–5181.[Abstract/Free Full Text]

  40. Fahling M, Steege A, Perlewitz A et al. Role of nucleolin in posttranscriptional control of MMP-9 expression. Biochim Biophys Acta 2005;1731:32–40.[Medline]

  41. Honda K, Ohno S, Tanimoto K et al. The effects of high magnitude cyclic tensile load on cartilage matrix metabolism in cultured chondrocytes. Eur J Cell Biol 2000;79:601–609.[CrossRef][Medline]

  42. Yang CM, Chien CS, Yao CC et al. Mechanical strain induces collagenase-3 (MMP-13) expression in MC3T3–E1 osteoblastic cells. J Biol Chem 2004;279:22158–22165.[Abstract/Free Full Text]

  43. Kurpinski K, Park J, Thakar RG et al. Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain. Mol Cell Biomech 2006;3:21–34.[Medline]

  44. Park JS, Chu JS, Cheng C et al. Differential effects of equiaxial and uniaxial strain on mesenchymal stem cells. Biotechnol Bioeng 2004;88:359–368.[CrossRef][Medline]

  45. Mott JD, Werb Z. Regulation of matrix biology by matrix metalloproteinases. Curr Opin Cell Biol 2004;16:558–564.[CrossRef][Medline]

  46. Parikka V, Vaananen A, Risteli J et al. Human mesenchymal stem cell derived osteoblasts degrade organic bone matrix in vitro by matrix metalloproteinases. Matrix Biol 2005;24:438–447.[CrossRef][Medline]

  47. Scutt A, Bertram P. Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells. Calcif Tissue Int 1999;64:69–77.[CrossRef][Medline]

  48. Liu Z, Xu J, Colvin JS et al. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 2002;16:859–869.[Abstract/Free Full Text]

  49. Fromigue O, Marie PJ, Lomri A. Bone morphogenetic protein-2 and transforming growth factor-beta2 interact to modulate human bone marrow stromal cell proliferation and differentiation. J Cell Biochem 1998;68:411–426.[CrossRef][Medline]

  50. Kalluri R. Basement membranes: Structure, assembly and role in tumour angiogenesis. Nat Rev Cancer 2003;3:422–433.[CrossRef][Medline]

  51. Heissig B, Hattori K, Dias S et al. Recruitment of stem and progenitor cells from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell 2002;109:625–637.[CrossRef][Medline]

  52. Inui T, Ishibashi O, Origane Y et al. Matrix metalloproteinases and lysosomal cysteine proteases in osteoclasts contribute to bone resorption through distinct modes of action. Biochem Biophys Res Commun 1999;258:173–178.[CrossRef][Medline]

  53. Song L, Webb NE, Song Y et al. Identification and functional analysis of candidate genes regulating mesenchymal stem cell self-renewal and multipotency. STEM CELLS 2006;24:1707–1718.[Abstract/Free Full Text]

  54. Rosowski M, Falb M, Tschirschmann M et al. Initiation of mesenchymal condensation in alginate hollow spheres—a useful model for understanding cartilage repair? Artif Organs 2006;30:775–784.[CrossRef][Medline]

  55. Li YJ, Batra NN, You L et al. Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation. J Orthop Res 2004;22:1283–1289.[CrossRef][Medline]

  56. Wong M, Siegrist M, Goodwin K. Cyclic tensile strain and cyclic hydrostatic pressure differentially regulate expression of hypertrophic markers in primary chondrocytes. Bone 2003;33:685–693.[Medline]

  57. Carter DR, Beaupre GS, Giori NJ et al. Mechanobiology of skeletal regeneration. Clin Orthop Relat Res 1998; (suppl 355)S41–S55.




This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
W. Zong, L. A. Meyn, and P. A. Moalli
The Amount and Activity of Active Matrix Metalloproteinase 13 Is Suppressed by Estradiol and Progesterone in Human Pelvic Floor Fibroblasts
Biol Reprod, February 1, 2009; 80(2): 367 - 374.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
2006-0676v1
2006-0676v2
25/8/1985    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kasper, G.
Right arrow Articles by Duda, G. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kasper, G.
Right arrow Articles by Duda, G. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS